TYPE Original Research
PUBLISHED 11 January 2023
DOI 10.3389/fphar.2022.1069321

OPEN ACCESSEDITED BY

Rong Wang,
People’s Liberation Army Joint Logistics
Support Force 940th Hospital, China
REVIEWED BY

Li Zhiling,
Shanghai Jiao Tong University, China
Mohammed E. El-Asrag,University of Birmingham, United Kingdom*CORRESPONDENCE

Masato Kobayashi,
kobayasi@mhs.mp.kanazawa-u.ac.jp
SPECIALTY SECTION

This article was submitted to Drug
Metabolism and Transport,a section of the journal
Frontiers in Pharmacology
RECEIVED 13 October 2022
ACCEPTED 29 December 2022
PUBLISHED 11 January 2023
CITATION

Sato K, Seki T, Mizutani A, Muranaka Y,
Hirota S, Nishi K, Yamazaki K, Nishii R,
Nakanishi T, Tamai I, Kawai K and
Kobayashi M (2023), A single high-dose
irradiation changes accumulation of
methotrexate and gene expression levels
of SLC and ABC transporters incancer cells.
Front. Pharmacol. 13:1069321.
doi: 10.3389/fphar.2022.1069321
COPYRIGHT© 2023 Sato, Seki, Mizutani, Muranaka,
Hirota, Nishi, Yamazaki, Nishii, Nakanishi,
Tamai, Kawai and Kobayashi. This is an
open-access article distributed under theterms of the Creative Commons
Attribution License (CC BY). The use,
distribution or reproduction in otherforums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication inthis journal is cited, in accordance withaccepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms.

A single high-dose irradiation
changes accumulation of
methotrexate and gene expression
levels of SLC and ABC transporters
in cancer cells
Kakeru Sato 1, Tatsuya Seki 1, Asuka Mizutani 2, Yuka Muranaka 1,
Shiho Hirota 1, Kodai Nishi 3, Kana Yamazaki 4, Ryuichi Nishii 4,
Takeo Nakanishi 5, Ikumi Tamai 6, Keiichi Kawai 2,7 andMasato Kobayashi 2*
1

Division of Health Sciences, Graduate School of Medical Sciences, Kanazawa University, Kanazawa, Japan,
Faculty of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University,
Kanazawa, Japan, 3Department of Radioisotope Medicine, Atomic Bomb Disease Institute, Nagasaki
University, Nagasaki, Japan, 4Department of Molecular Imaging and Theranostics, Institute for Quantum
Medical Science, Quantum Life and Medical Science Directorate, National Institutes for Quantum Scienceand Technology, Chiba, Japan, 5Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki,
Gunma, Japan, 6Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health
Sciences, Kanazawa University, Kanazawa, Japan, 7Biomedical Imaging Research Center, University of Fukui,
Fukui, Japan
2

Chemoradiotherapy is frequently used to treat cancer. Stereotactic bodyradiotherapy (SBRT) is a single high-dose radiotherapy used to treat a variety ofcancers. The anticancer drug methotrexate (MTX) shows afﬁnity for solute carrier(SLC) and ATP-binding cassette (ABC) transporters. This study investigatedrelationships between accumulation of methotrexate and gene expression levelsof solute carrier and ATP-binding cassette transporters in cancer cells after a single
and high-dose X-ray irradiation. Cancer cell lines were selected from lung andcervical cancer cell line that are commonly used for stereotactic body radiotherapy
and effective with methotrexate. We examined expression levels of organic aniontransporting polypeptide (OATP)1B1, OATP1B3, OATP1B7, and organic aniontransporter (OAT)1 as solute carrier transporters and multidrug resistanceassociated protein (MRP)1 and MRP2 as ATP-binding cassette transporters, using
real-time polymerase chain reaction and accumulation of 3H-MTX in cancer cells
after 10-Gy irradiation, assuming stereotactic body radiotherapy. Cells were divided
into three groups: Control without irradiation; 4 h after irradiation; and 24 h after
irradiation. In control, gene expression levels of OAT1 in all cells was below the limitof measurement. After irradiation, gene expression levels of OATP1B1/1B3/
1B7 showed changes in each cell line. Gene expression levels of MRP1/2 tended
to increase after irradiation. Gene expression levels of OATP1B1/1B3/1B7 were much
lower than those of MRP1/2. Accumulation of 3H-MTX tended to decrease over timeafter irradiation. Irradiation of cancer cells thus alters gene expression levels of both
solute carrier transporters (OATP1B1/1B3/1B7) and ABC transporters (MRP1/2) anddecreases accumulation of 3H-MTX in cancer cells over time due to elevatedexpression of MRP1/2.
KEYWORDS

chemoradiotherapy, methotrexate, SLC transporter, ABC transporters, stereotactic bodyradiotherapy, x-ray, irradiation

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1 Introduction

Chemicals) and Eagle’s minimum essential medium (FUJIFILM
Wako Chemicals) mixed with 10% fetal bovine serum and 1%
sodium pyruvate at 37°C under conditions of 5% CO2.

Chemoradiotherapy combines chemotherapy with anticancer
drugs and radiotherapy to treat cancer. Chemoradiotherapy can bedivided into three categories according to the timing of anticancer
drug administration: Neoadjuvant; concurrent; and adjuvant (Baldini
et al., 2018). The accumulation of anticancer drugs in cancer cells
usually depends on gene expression levels of solute carrier (SLC) andATP-binding cassette (ABC) transporters (Nakanishi, 2007;
Carmichael and Day, 2022). SLC transporters mainly contribute to
the uptake of anticancer drugs, while ABC transporters are involved intheir excretion (Nakanishi, 2007; Carmichael and Day, 2022).
In radiotherapy, stereotactic radiotherapy involves the delivery ofhigher doses (7–18 Gy or more) than the usual single-beam dose
(1.8–2 Gy) and is used to treat various cancers (Marcrom et al., 2017;
Jardel et al., 2020; Sarudis et al., 2021; Ugurluer et al., 2021).
Stereotactic radiotherapy was originally used to treat brain cancers,
with stereotactic body radiotherapy (SBRT) representing theapplication of this technology to the trunk of the body, such as forlung and liver cancers (Song et al., 2004; Donovan and Swaminath,
2018; Tandberg et al., 2018; Sarudis et al., 2021; Ugurluer et al., 2021).
SBRT has also been shown to be effective against cervical cancer,
which is still frequently treated using intracavitary small-source
radiotherapy (Ito et al., 2019; Facondo et al., 2021)
Methotrexate (MTX) is a folate antagonist used as an anticancer
drug (Visentin et al., 2012). This agent stops cancer growth by
preventing the uptake of folic acid, which is necessary in DNA
synthesis (Yu et al., 2020). MTX is effective against lung andcervical cancers, where SBRT also appears useful (Conroy et al.,
1976; Smyth and Ford, 1981). MTX has shown afﬁnity for the SLC
transporters organic anion-transporting polypeptide (OATP) andorganic anion transporter (OAT), and the ABC transporters
multidrug resistance-associated protein (MRP), multidrug
resistance protein (MDR), and breast cancer resistance protein(BCRP) (Hagenbuch and Meier, 2004; Nakanishi, 2007; Murakami
and Mori, 2012; Gao et al., 2021). While irradiation increases theexpressions of MRP1 and MRP2, contributing to the efﬂux of MTX(Henness et al., 2002; Bartkowiak et al., 2009), the effects of irradiation
on SLC transporters have not been examined. Further, correlations
between the kinetics of anticancer drugs including MTX and SLC and
ABC transporters after irradiation have yet to be clariﬁed. The purposeof this study was thus to investigate the relationships between
accumulation of MTX and expression levels of the genes for SLC
and ABC transporters in cancer cells after irradiation. Temporalchanges in MTX accumulation in cancer cells after a single andhigh-dose irradiations assuming SBRT were examined.

2.2 Irradiation of cell lines
After achieving 70–80% conﬂuence in a 10-cm diameter plate,each cell line was irradiated with a single 10-Gy X-ray (dose rate,
1.0 Gy/min) using X-ray irradiation equipment (MBR1520R-3;
Hitachi, Tokyo, Japan). Cells were divided into three groups:
Control without irradiation; 4 h after irradiation; and 24 h after
irradiation.

2.3 RNA extraction and quality assessment
An RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany) was used
to extract RNA from the cancer cells used in this study. The quality ofthe extracted RNA was evaluated using the RNA integrity number(RIN) as an indicator of quality. The RIN is expressed as a number
from 1 to 10, with a higher number reﬂecting higher quality of RNA.
An Afﬁnity Script QPCR cDNA Synthesis kit (Agilent Technologies,
Tokyo, Japan) was used for synthesizing cDNA.

2.4 Conducting real-time polymerase chain
reaction (PCR)
Real-time PCR was performed using an AriaMx 5P system(Agilent Technologies). OATP1B1/1B3/1B7 as the combination of
OATP1B1, OATP1B3 and OATP1B7 for SLC transporters (because
the primer sequences of these transporters are quite similar) and
MRP1 and MRP2 for ABC transporters were selected as the targets of
PCR. The gene ACBT for β-actin was used as the internal control gene,as a housekeeping gene that is constantly expressed in all cells. Also,
ACBT was used to correct for differences in the amounts of initial
RNA and cDNA due to differences in sample organization andpuriﬁcation methods. Primer design was outsourced to Euroﬁns
Genomics (Tokyo, Japan). Preparation of cloned plasmids used forthe creation of standard curves was outsourced to GenScript (Tokyo,
Japan). Primer sequences and concentrations of the genes used areshown in Table 1. A 20-µL volume of PCR reaction solution contained
10 µL of Brilliant III Ultra-Fast SYBR Green QPCR Master Mix(Agilent Technologies), .4 µL of primer, 1 µL of template (10–50 ng
of cDNA or cloned plasmid) and 8.6 µL of nuclease-free water. The
thermal proﬁle of reaction conditions was: 95°C hot start for 3 min,then 45 cycles of ampliﬁcation at 95°C for 5 s and 62°C for 15 s, ending
with 95°C for 1 min, 55°C for 30 s and 95°C for 30 s.

2 Material and methods
2.1 Cancer cell lines

2.5 Accumulation of 3H-MTX in cancer cells

The human-derived lung adenocarcinoma cancer cell lines NCIH441 (American Type Culture Collection, Manassas, VA,
United States) and PC-14 (RIKEN Cell Bank, Tsukuba, Japan)
were used. The HeLa human-derived cervical cancer line (RIKENCell Bank) was also used. H441, PC-14 and HeLa cell lines were
cultivated using RPMI-1640 (FUJIFILM Wako Chemical, Osaka,
Japan), Dulbecco’s Modiﬁed Eagle’s Medium (FUJIFILM Wako

Frontiers in Pharmacology

Each cell was seeded at 1.0 ×105 cells/well in 12-well plastic plates.
At about 24 h after seeding, cells were irradiated and divided into threegroups: 4 h after irradiation; 24 h after irradiation; and control without
irradiation. Each group was pre-incubated for 5 min in phosphatebuffered saline (PBS), then incubated with 3H-MTX (10 kBq/well) for
5, 10, 30, or 60 min. After incubation, cells were washed twice with

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TABLE 1 Primer sequences and concentrations of the used genes.

Transporters/Housekeeping
gene
OATP1B1/1B3/1B7

Gene symbol

Primer sequence

Concentration (nM)

Forward

ReverseOATP1B1

SLCO1B1

GCACTGGGTTTCCACTCAATCAGTTGTTGGTGGACCACTTTOATP1B3

SLCO1B3GCAATGGGTTTCCAGTCAATAGCTGTTGGTGGACCACTTCOATP1B7

SLCO1B7GCAATCGGCTTCCATTCAATAGCTGTTGGTGGACCACTTCOAT1SLC22A6GCGCCTTTTTTTGCCTTCTTTCCCGCTTCCCATTGATC

300

MRP1

ABCC1

GACCATGAATGTGCAGAAGGGCCTCATCCAACACAAGGAT

100

MRP2

ABCC2

CTGCGGCTCTCATTCAGTCTGCCAAGTTGGATAGGGTCAA

ACTB

ACTB

CCAACCGCGAGAAGATGACCAGAGGCGTACAGGGATAG

HeLa cells, but OAT1 in all cells was below the limit ofmeasurement. After cell irradiation, total gene expression levels of
OATP1B1/1B3/1B7 decreased in H441 in comparison to before
irradiation (control), but increased slightly in PC-14 and HeLa cells.
For ABC transporters, expression levels of MRP1/2, as the
combination of MRP1 and MRP2, were higher than levels of
OATP1B1/1B3/1B7 and OAT1 in control samples of all cancer
cells. In control samples, MRP1/2 showed higher expression in
HeLa than in H441 or PC-14. In addition, H441 and PC-14
showed higher gene expression levels of MRP1 than MRP2, while
HeLa displayed higher gene expression levels of MRP2 than MRP1.
After irradiation, gene expression levels of MRP1/2 tended to increaseover time in all cancer cell lines.
Figure 1 shows the accumulation of 3H-MTX in H441, PC-14 andHeLa cells in the control, 4 h after irradiation and 24 h after irradiationgroups at 5, 10, 30, and 60 min after 3H-MTX injection. Accumulationof 3H-MTX was decreased at 4 and 24 h after irradiation in H441 cells
and at 24 h after irradiation in PC-14 cells. In HeLa cells, accumulationof 3H-MTX was signiﬁcantly decreased compared to control from10 min after 3H-MTX injection in the 4 h after irradiation group andfrom 30 min after 3H-MTX injection in the 24 h after irradiationgroup.

TABLE 2 Gene expression levels of measured drug transporter in each cell byconducting Real-time PCR.

Gene expression
level (×105)
Transporter

Condition

H441

PC-14

HeLa

OATP1B1/1B3/1B7

Control

13.0

0.01

0.04

4 h after irradiation

10.5

0.05

0.06

24 h after irradiation

10.0

0.04

0.09

Control

64.5

56.1

34.5

4 h after irradiation

50.3

78.9

51.0

24 h after irradiation

79.3

90.3

70.8

Control

0.30

1.80

56.7

4 h after irradiation

0.40

2.30

60.2

24 h after irradiation

0.80

2.70

98.0

Control

64.8

57.9

91.2

4 h after irradiation

50.7

81.2

111.2

24 h after irradiation

80.1

93.0

168.8

MRP1

MRP2

MRP1/2200

4 Discussion
600 µL of PBS and lysed by 500 µL of .1 M NaOH. Three hundred
microliters of cell lysate were mixed with 5 mL of ULTIMA GOLD
(Perkin Elmer, Waltham, MA, United States) and the radioactivity ofthe mixture was measured using a liquid scintillation counter (LSC5100; Hitachi Aloka Medical, Tokyo, Japan). The results are shown as
the percent injected dose/number of living cells measured by anautomatic cell counter (LUNA FX7 ; Logo Biosystems, Gyeonggido, South Korea).

Consideration of the effects of irradiation on the kinetics ofanticancer drugs in chemoradiotherapy is important. This study
examined how the accumulation of 3H-MTX in cancer cells was
impacted by the effects of gene expression levels for SLC and ABC
transporters after X-ray irradiation. Since the degree to which gene
expression levels of SLC and ABC transporters are changed under theinﬂuence of irradiation was unknown, we examined these gene
expressions after irradiation by conducting Real-time PCR(Table 2). Total gene expression levels of OATP1B1/1B3/
1B7 decreased after irradiation in H441 cells compared to control,
but increased slightly in PC-14 and HeLa cells. For ABC transporters,
total gene expression levels of MRP1/2 were higher than OATP1B1/
1B3/1B7 and OAT1 in all cancer cell lines under the control
conditions. Accumulation of 3H-MTX tended to decrease over timeafter irradiation in all cancer cell lines (Figure 1), and accumulation
was signiﬁcantly decreased at 24 h.

™

3 Results
All cell lines showed RIN >9, indicating high-quality RNA.
Measured expression levels of drug transporter genes in each cellline in the three groups by conducting Real-time PCR are shown in
Table 2. For SLC transporters, the total gene expression level of
OATP1B1/1B3/1B7 was higher in H441 cells than in PC-14 orFrontiers in Pharmacology

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et al. (2002) reported that expressions of MRP1 and MRP2 wereincreased after fractionated irradiation, but these expression levels
might change over time after a single high-dose irradiation. In PC14 and HeLa cells (Table 2), gene expression levels of MRP1/2 weremuch greater than those of OATP1B1/1B3/1B7. After irradiation,
the difference between OATP1B1/1B3/1B7 and MRP1/2 became
greater over time.
In PC-14 (Figure 1B), accumulation of 3H-MTX showed littlechange between control and 4 h after irradiation, but wassigniﬁcantly decreased at all time points after adding 3H-MTX incells at 24 h after irradiation. Since gene expression levels of
OATP1B1/1B3/1B7 were slightly greater in PC-14 cells, theeffects on gene expression levels of OATP1B1/1B3/1B7 may begreater than the effects on accumulation of 3H-MTX at 4 h after
irradiation. At 24 h after irradiation, a correlation was noted between
decrease in accumulation of 3H-MTX and much higher gene
expression levels of MRP1/2.
In HeLa cells (Figure 1C), accumulation of 3H-MTX wassigniﬁcantly decreased compared to control at 4 h after
irradiation from 10 min after adding 3H-MTX, and at 24 h after
irradiation from 30 min after adding 3H-MTX. Although theeffects of drug transporters are usually seen at 5 min after
adding 3 H-MTX, no signiﬁcant differences at this time points
were seen between control and groups at 4 and 24 h after
irradiation. An equilibrium state appears to exist between
functions of OATP1B1/1B3/1B7 and MRP1/2 at around 5 minafter adding 3H-MTX, but gene expression levels of MRP1/
2 were higher than those of OATP1B1/1B3/1B7 (Table 2). With
greater expression of MRP1/2 over time, accumulation of 3H-MTX
decreased signiﬁcantly compared to control (Figure 1C). From
10 min after adding 3H-MTX, accumulation of 3H-MTX was
higher in HeLa than in H441 and PC-14. Gene expression
levels of MRP2 were also higher in HeLa than in H441 or PC14, and expression of MRP2 was also higher than that of MRP1.
These results may suggest that 3 H-MTX has higher afﬁnity for
MRP1 than for MRP2.
This study was performed assuming SBRT, in which a single
exposure provides a higher dose than conventional radiotherapy(Marcrom et al., 2017; Jardel et al., 2020; Sarudis et al., 2021;
Ugurluer et al., 2021). Since Lei et al. (2021) reported that thesurvival rate of HeLa was less than 50% after a single 10-Gy
irradiation, we selected a single high-dose of 10-Gy irradiation. In
our experiments, cancer cells after a single high-dose irradiation
have shown a tendency to excrete anticancer drugs as a foreign body.
Neoadjuvant chemotherapy, which administers anticancer drugs
prior to radiation, may therefore prove effective in the
combination of SBRT and chemotherapy. However, these results
only reﬂect temporal changes following a single irradiation. Future
experiments will need to consider fractional irradiation at high dose.
In addition, in vivo experiments will be required to conﬁrm ourin vitro results for the accumulation of 3H-MTX and expression of
drug transporters.
For a more detailed examination, next-generation sequencers
might be useful in the future because this method is capable ofcomprehensively quantifying multitude of various genes (Slatko
et al., 2018). However, we intentionally selected MTX which has
afﬁnity primarily for OATP and MRP transporters as an anticancer drug. Therefore, Real-time PCR, which can accurately
quantify gene expression levels of speciﬁc transporters, would be

FIGURE 1
Accumulation of 3H-MTX in H441 (A), PC-14 (B) and HeLa (C) cells
in the three groups including control, 4 h after irradiation and 24 h after
irradiation at 5, 10, 30, and 60 min after adding 3H-MTX. Accumulation of
3
H-MTX tended to decrease after irradiation in all three cancer cells.
†
p < .01 and *p < .05 vs. control.

The correlation between accumulation of 3H-MTX and gene
expression levels of drug transporters is discussed for each cell line.
In H441 cells (Table 2), gene expression levels of OATP1B1/1B3/
1B7 decreased over time, and MRP1/2 was slightly decreased at 4 hafter irradiation in comparison with control and increased further
at 24 h after irradiation. Accumulation of 3H-MTX in H441 cells
was signiﬁcantly decreased compared to control at 5, 10, and30 min after adding 3H-MTX (Figure 1A). Although we selected
MRP1/2 as representative ABC transporters for MTX, changes in
MDR and BCRP gene expressions might also inﬂuence theaccumulation of 3H-MTX in H441 (Ji et al., 2013). Henness

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Funding

appropriate in this study. The Real-time PCR was also used in the
study of (Sutherland et al., 2020). They examined the relationshipbetween accumulation of anti-cancer drugs in cancer cells and gene
expression levels of speciﬁc SLC transporters by Real-time PCR.

This study was funded in part by Grants-in-Aid for ScientiﬁcResearch from the Japan Society for the Promotion of Science (Nos
21H02865, 22H03016, and 22K19504) and the Network-type
Joint Usage/Research Center for Radiation Disaster Medical
Science.

5 Conclusion
X-ray irradiation with a single, high dose to cancer cells alters gene
expression levels of both SLC transporters (OATP1B1/1B3/1B7) and
ABC transporters (MRP1/2). In particular, changes in MRP1/2 weremuch greater than those in OATP1B1/1B3/1B7. Irradiation decreased
accumulation of 3H-MTX in cancer cells over time due to higher
expression of MRP1/2.

Acknowledgments
The authors would like to thank Mikie Ohtake and other staff ofthe Faculty of Health Sciences, Kanazawa University.

Conﬂict of interest

Data availability statement

The authors declare that the research was conducted in theabsence of any commercial or ﬁnancial relationships that could beconstrued as a potential conﬂict of interest.

The original contributions presented in the study are included in
the article/supplementary materials, further inquiries can be directedto the corresponding author.

Publisher’s note

Author contributions

All claims expressed in this article are solely those of the authorsand do not necessarily represent those of their afﬁliatedorganizations, or those of the publisher, the editors and thereviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.

Conceptualization, MK; methodology, KS, TS, and SH; investigation,
KS, TS, AM, SH, YM, KN, and MK; resources, KY and RN;
writing—original draft preparation, KS; writing—review and editing,TN, IT, KK, and MK; supervision, TN, IT, KK, and MK. All authors
have read and agreed to the published version of the manuscript.

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